Various organisms have at least one enzyme that degrades the RNA of RNA-DNA hybrids. Such hybrids result in vivo from transcription and often are associated with DNA replication, even in replication of retroviruses including HIV. These ribonucleases H (RNases H), so far, fall into two classes based upon primary amino acid sequence similarity. From our studies, we know that the well-characterized Escherichia coli RNase HI has homologs in many different species including human and mouse. We know that these mammalian proteins resemble in sequence and function the RNase H1 of Saccharomyces cerevisiae by having a double-stranded RNA-binding activity in addition to the RNase H activity. Similarly, the bacterial RNase HII protein has counterparts in eukaryotes. RNases H perform important cellular functions and it is important to know how and by what means these enzymes are synthesized. For example, the use of DNA drugs employed in oligonucleotide-based antisense therapy relies on endogenous RNases H to degrade certain disease causing mRNAs (RNAs synthesized at inappropriate times or locations). The ability to increase RNase H activity in target cells could make these antisense DNAs more effective drugs. In a similar vein, certain types of drugs targeted to inhibit the RNase H activity of HIV reverse transcriptase could also inhibit the cellular enzymes leading to undesired effects. The RNase H2Ap protein purified from S. cerevisiae has high levels of RNase H activity yet the same protein expressed in E. coli is inactive. We find other polypeptides co-purify with the active form of the enzyme suggesting that multiple subunits comprises the active enzyme. Cell-cycle regulation of the RNH2A gene expression together with the differential expression due to the overlapping DNA sites suggests the protein may have different subunits at different stages of the cell-cycle to participate in either DNA replication or DNA repair. These results indicate induction or expression of the RNase H2Ap may not be sufficient for increasing RNase H activity associated with this polypeptide. In fact, when overexpression of the RNase H2Ap does occur, we have found only a modest increase in RNase H activity. We have also found that the RNase H1 protein of human and mouse form a dimer in the presence of substrate and that this complex leads to processivity of the enzyme (i.e., the protein tends to degrade one molecule of substrate regradless of size before releasing and attacking a second molecule). These results from this year help us to understand the roles mechanism of action of these important proteins